On 9 Nov 2005 06:37:19 -0800, "vijaykr" <vijaykr at gmail.com> wrote:
>Yesterday, I did the miniprep and EcoRI digestion. Its pretty clear
>that the plasmids are not having insert. There was a single band that
>corresponded to the size of the vector.
I would suggest that you sequence the plasmid you get from the
miniprep. From your description alone, the ligation must have
worked, although it is difficult to tell here from afar if you have
done anything wrong that might have caused the problem (it is always
easier to see someone actually doing it to identify any problem).
The insert may have deleted itself by recombination (so sequencing is
useful for checking), so check and compare the sequence of both your
insert and the plasmid to see if there is any possibility of that. A
very unlikely possibility is the presence of intein in your insert
which can somehow cause the problem. Someone I know had a problem
with an intein which he didn't know was present (he solved the problem
by identifying and removing the intein sequence), but I don't know if
intein can cause the deletion your insert.
If your insert is large and the plasmid is high copy number, a useful
way of doing screening for insert is to pick the colonies from plate,
lyse the cells and run the cell lysate in the gel. You then look for
size difference between the plasmids with insert and the one without
(run one without insert). In your case this method might be useful
and you can screen a large number relatively easily and quickly.
There are many protocols around for this screening method, have a look
in Google, if you can't find them, let me know.
Try directional cloning and see if it makes any difference.
>As a control to check EcoRI activity I transformed the EcoRI cut vector
>after column purifying without ligating and another control to check
>CIP activity by religating CIP treated vector and transformed this one
>also. The former control gave three colonies and the later one showed 7
>colonies.